Abstract
Receptor tyrosine phosphatase sigma (RPTPσ) plays an important role in the regulation of axonal outgrowth and neural regeneration. Recent studies have identified two RPTPσ ligands, chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPG), which can modulate RPTPσ activity by affecting its dimerization status. Here, we developed a split luciferase assay to monitor RPTPσ dimerization in living cells. Using this system, we demonstrate that heparin, an analog of heparan sulfate, induced the dimerization of RPTPσ, whereas chondroitin sulfate increased RPTPσ activity by inhibiting RPTPσ dimerization. Also, we generated several novel RPTPσ IgG monoclonal antibodies, to identify one that modulates its activity by inducing/stabilizing dimerization in living cells. Lastly, we demonstrate that this antibody promotes neurite outgrowth in SH-SY5Y cells. In summary, we demonstrated that the split luciferase RPTPσ activity assay is a novel high-throughput approach for discovering novel RPTPσ modulators that can promote axonal outgrowth and neural regeneration.
Highlights
Deregulation of protein tyrosine phosphorylation impacts a broad spectrum of human diseases including obesity, vascular diseases, cancers and neural degeneration [1, 2]
We observed a correlation between the expression levels of the extracellular domain of RPTPσ and its luminescence signal when compared to full length RPTPσ
RPTPσ dimerization is not due to transient overexpression artifact since the DIDII domain of RPTPσ had low luminescence activity. This result indicates that RPTPσ dimers formation are driven by the presence of pre-existing extracellular ligands including the previously reported RPTPσ ligands, heparan sulfate proteoglycans (HSPG) which is known to be expressed on Hek293T cell surface [23]
Summary
Deregulation of protein tyrosine phosphorylation impacts a broad spectrum of human diseases including obesity, vascular diseases, cancers and neural degeneration [1, 2]. Receptor protein tyrosine phosphatase sigma (RPTPσ) is highly expressed throughout neural development and remains important for neural plasticity even in the adult brain [3,4,5,6,7]. Extracellular matrix proteins CSPGs (chondroitin sulfate proteoglycans) and HSPGs (heparan sulfate proteoglycans) were identified as ligands of RPTPσ, suggesting a potential mechanism of RPTPσ regulation in nerve injury models [4, 6, 7]. These studies have demonstrated that binding of CSPG to RPTPσ receptors expressed on the axon surface induces axonal retraction [4, 6]. Several synaptic molecules including TrkC, Netrin-G3, and Slitrk have been
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